The overall goal of this exploratory research project is to conduct a proteomic analysis of the vaccinia virus (VV) virion, which will provide the foundation for the development of new antiviral intervention strategy and prevention programs to prevent diseases caused by orthopoxviruses such as smallpox. The VV genome has been sequenced since 1990 (1), but the roles of about half of the genes have remained entirely unknown. Although, numerous proteins of vaccinia virus have been studied in great detail, there no comprehensive proteomic analysis of the VV particle is available. To advance our understanding of the replication cycle of the VV and other more pathogenic members of the orthopoxvirus family and to discover proteins that may become targets for a new antiviral drugs or antigens for a new generation of vaccines we define two specific aims for this research project: 1.) Establish virion-specific proteome reference catalogs for the intracellular mature virus (IMV) by using mass spectrometry; 2.) Perform proteomic analysis of post-translational modifications (PTMs) present on virion proteins, focusing initially on phosphoproteins and acylproteins. It is expected that the proteome analysis of VV will yield information on the expression and location of new viral gene products. Future efforts can then focus on their isolation and in-depth characterization. The availability of comprehensive proteome reference maps of the VV virion forms will be invaluable for poxvirologists and will provide key data for novel prevention and treatment strategies for viral diseases.